1. Field of the Invention
This invention is directed to papovavirus-derived episomes that replicate efficiently in mammalian cells, yielding stable transfectants having a high episomal copy number and expressing encoded genes at high levels. Papovavirus-derived episomes may be useful in gene therapy strategies to modulate the growth of bladder carcinoma cells.
2. Review of Related Art
One approach to gene therapy of human cancer cells is to introduce vectors expressing antisense sequences to block expression of dominant oncogenes and growth factor receptors. However, high-level expression of the oncogenes requires comparable levels of antisense expression, which presents a considerable technical obstacle, particularly when using expression vectors having a limited potential for achieving multiple copies in stable transfectants. Human cells transduced by retroviral vectors have only one or several copies of integrated retrovirus in stable transfectants. In contrast, hundreds of copies of episomal plasmids can accumulate in stable transfectants because these vectors replicate extrachromosomally. One method to express high levels of antisense transcripts is to utilize episomal plasmid vectors than can replicate extrachromosomally in human cells.
Attempts to produce episomal vectors that will replicate in some types of human cells are reported by the literature. Episomal plasmids have been developed from several DNA viruses, including bovine papilloma virus (BPV) (Sarver, et al., 1981, Mol. Cell. Biol., 1: 486-496; DiMaio, et al., 1982, Proc. Natl. Acad. Sci., U.S.A., 97: 4030-4034), SV40 (Tsui, et al., 1982, Cell, 30: 499-508), Epstein-Barr virus (EBV) (Yates, et al., 1985, Nature, 313: 812-815; Margolskee, et al., 1988, Mol. Cell. Biol., 8: 2837-2847; Belt, et al., 1989, Gene, 84: 407-417; Chittenden, et al., 1989, J. Virol., 63: 3016-3025), and BK virus (BKV) (Milanesi, et al., 1984, Mol. Cell. Biol., 4: 1551-1560). Each of these episomal plasmids contains a viral origin of DNA replication and a virally encoded early gene that trans-activates the viral origin and allows the episome to replicate in the transfected host cell.
Although EBV-based episomes have been used to efficiently screen cDNA libraries, the EBV system has limited applications to non-lymphoid cell types (Vidal, et at., 1990, Biochim. Biophys. Acta 1048: 171-177)), and the EBV replicon is not active in many cell types. Additionally, EBNA-1 is one of several EBV latent genes that immortalize human lymphocytes, and transfection of the EBV-negative BJAB lymphoma cell line by EBNA-1 induces soft agar growth, indicating transformation of the cells. (Konoshita, 1990, Hokkaido Igaku Zasshi, 65: 362-375)
Furthermore, stable transfection efficiencies for EBNA-1 negative cell lines transduced by EBV episomal plasmids encoding EBNA-1 (transactivator) and ORI-P (EBV DNA origin) are low, not significantly better than non-episomal plasmids (Yates, et al., 1985; Vidal, et at., 1990. However, if EBNA-1 is expressed in cells prior to transfection, then a subsequent transfection with a plasmid containing ORI-P and a selectable marker can yield stable transfection efficiencies of up to 10% (Margolskee, et al., 1988; Belt, et al., 1989; Yates, et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81: 3806-3810; Lutfallia, et al., 1989, Gene 76: 27-39). Comparable results have been noted in a related system of COS cell clones expressing high levels of SV-T, which permit efficient replication of SV40 origin-containing plasmids in transient transfectants (Tsui, et al., 1982; Rio, et al., 1985, Science 227: 23-28; Chittenden, et al., 1991, J. Virol. 65: 5944-5951).
In the COS cell system, however, episomal replication can proceed in a runaway fashion, resulting in up to 10.sup.4 episomal copies by 48 hours after transfection. Despite efficient episomal replication in transient transfectants, low stable transfection efficiencies have been noted in these studies (Chittenden, et al., 1991; Roberts, et al., 1986, Cell, 46: 741-752). Presumably, most transient transfectants die secondary to episome-mediated cell death (Chittenden, et al., 1991; Roberts, et al., 1986).
However, transfection of COS cells by SV40 DNA origin-containing plasmids does produce stable transfectants having episomal plasmids (Tsui, et al., 1982), and it may be possible to control runaway episomal replication by a variety of strategies, including use of replication control regions from other viruses. For example, runaway episomal replication in COS cell clones can be controlled by use of plasmids containing the SV40 DNA origin and regions of the bovine papilloma virus (BPV) replicon (Roberts, et al., 1986; Roberts, et al., 1988, Cell 52: 397-404). These studies have identified two BPV sequences (NCOR I and NCOR II) that modulate runaway SV40 episomal replication in transient transfectants, and a third trans-suppressing factor encoded by 5' sequences in the E1 open reading frame. Hybrid plasmids encoding the SV40 DNA origin and a 2113 bp EcoR1 fragment of BPV have substantially higher stable transfection efficiencies than pSV-NEO (Roberts, et al., 1986). A DNA homology search failed to identify similar NCOR sequences in the BKV or SV40 replicon.
Thus, there remains a need for vectors which will replicate episomally in a controlled fashion in mammalian cells for gene therapy applications. In particular, there is a need for vectors that will replicate episomally in human cells without transforming the cells.